Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2
Alexey Bersenev, … , Joanna Balcerek, Wei Tong
Alexey Bersenev, … , Joanna Balcerek, Wei Tong
Published July 10, 2008
Citation Information: J Clin Invest. 2008;118(8):2832-2844. https://doi.org/10.1172/JCI35808.
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Research Article Hematology

Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2

Abstract

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk–/– mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk–/– HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.

Authors

Alexey Bersenev, Chao Wu, Joanna Balcerek, Wei Tong

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Figure 5

The Lnk SH2 domain associates with kinase-active JAK2.

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Lnk constitutively associates Mpl. We established stable 32D-B/A cell li...
(A) Stable 32D-B/A cells expressing Mpl and Flag-tagged Lnk, in conjunction with either myc-tagged WT JAK2, or kinase-inactive JAK2 mutant Y1007F/Y1008F, were starved and stimulated with TPO for 10 minutes. Cell lysates were then precipitated with Flag-specific antibodies (left panel) and blotted with antibodies specific for myc (top), phosphotyrosine 4G10 (middle), or Lnk (bottom). The right panels show precipitation with myc-specific antibodies and blots with 4G10-specific (top) or myc-tagged antibodies (bottom). (B) We established stable 32D-B/A cell lines expressing Mpl along with Flag-tagged WT Lnk or various Lnk mutants with individual functional domains (R364E, W191A, and Y536F) abolished. Cells were starved and stimulated with TPO. Cell lysates were precipitated with Flag-specific antibodies (left panel) and blotted with antibodies specific for JAK2 (top) or Lnk (bottom). The right panels show precipitation with JAK2-specific antibodies and blots with antibodies specific for 4G10 (top) and JAK2 (bottom). C, control parental 32D-B/A cells; IgG(H), IgG heavy chain.